title: Unique structure and dynamics of the EphA5 ligand binding domain mediate its binding specificity as revealed by X-ray crystallography, NMR and MD simulations.
abstract: The 16 EphA and EphB receptors represent the largest family of receptor tyrosine kinases, and their interactions with 9 ephrin-A and ephrin-B ligands initiate bidirectional signals controlling many physiological and pathological processes. Most interactions occur between receptor and ephrins of the same class, and only EphA4 can bind all A and B ephrins. To understand the structural and dynamic principles that enable Eph receptors to utilize the same jellyroll β-sandwich fold to bind ephrins, the VAPB-MSP domain, peptides and small molecules, we have used crystallography, NMR and molecular dynamics (MD) simulations to determine the first structure and dynamics of the EphA5 ligand-binding domain (LBD), which only binds ephrin-A ligands. Unexpectedly, despite being unbound, the high affinity ephrin-binding pocket of EphA5 resembles that of other Eph receptors bound to ephrins, with a helical conformation over the J-K loop and an open pocket. The openness of the pocket is further supported by NMR hydrogen/deuterium exchange data and MD simulations. Additionally, the EphA5 LBD undergoes significant picosecond-nanosecond conformational exchanges over the loops, as revealed by NMR and MD simulations, but lacks global conformational exchanges on the microsecond-millisecond time scale. This is markedly different from the EphA4 LBD, which shares 74% sequence identity and 87% homology. Consequently, the unbound EphA5 LBD appears to comprise an ensemble of open conformations that have only small variations over the loops and appear ready to bind ephrin-A ligands. These findings show how two proteins with high sequence homology and structural similarity are still able to achieve distinctive binding specificities through different dynamics, which may represent a general mechanism whereby the same protein fold can serve for different functions. Our findings also suggest that a promising strategy to design agonists/antagonists with high affinity and selectivity might be to target specific dynamic states of the Eph receptor LBDs.
authors.affiliation: NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore, Republic of Singapore.
authors.forename: Xuelu
authors.initials: X
authors.lastname: Huan
authors.forename: Jiahai
authors.initials: J
authors.lastname: Shi
authors.forename: Liangzhong
authors.initials: L
authors.lastname: Lim
authors.forename: Sayantan
authors.initials: S
authors.lastname: Mitra
authors.forename: Wanlong
authors.initials: W
authors.lastname: Zhu
authors.forename: Haina
authors.initials: H
authors.lastname: Qin
authors.forename: Elena B
authors.initials: EB
authors.lastname: Pasquale
authors.forename: Jianxing
authors.initials: J
authors.lastname: Song
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D017931:DNA Primers; D006801:Humans; D008024:Ligands; D056004:Molecular Dynamics Simulation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D036123:Receptor, EphA5 / Q000737:chemistry / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D008024:Ligands; C484173:EPHA5 protein, human; D036123:Receptor, EphA5